Back-splicing Primers-based PCR Method for Specific Detection of circRNA

doi:10.13560/j.cnki.biotech.bull.1985.2021-0746

Abstract

Abstract:

In order to specifically amplify the contained circRNA in alternative back-splicing circularization events and accurately analyze the expression level of the target circRNA,we firstly analyzed the characteristics of the alternative back-splicing circularization events of high-confidence circRNA in grape（Vitis vinifera L.）through bioinformatics,and further proposed an improved method of primers design suitable for the alternative back-splicing circularization events,that is,the 3' end of one primer spanned 3-4 bases of the back-splicing junction. Moreover,we verified it via RT-PCR and qRT-PCR. The results showed alternative back-splicing circularization events were found in 21.7% of the source genes of high-confidence grape circRNA,which could be divided into juxtaposition,crossover and inclusion relationships. We selected 4 groups of circRNA for amplification,circRNA_4363,circRNA_6017,circRNA_6044 and circRNA_7086 were contained by circRNA_4364,circRNA_6018,circRNA_6045 and circRNA_7085,respectively. Two amplified bands were obtained by RT-PCR of the 4 contained circRNAs with conventional divergent primers,and the qRT-PCR dissolution curve was bimodal. A single amplified product can be obtained by using modified primers to perform RT-PCR and qRT-PCR on circRNA_4363,circRNA_6017 and circRNA_7086. Compared with conventional divergent primers,using modified primers specifically amplified the contained circRNA in alternative back-splicing circularization events,making the quantitative analysis results more accurate and reliable.

Key words: circRNA, qRT-PCR, divergent primer, alternative back-splicing

SUN Bao-zhen, QUAN Long-ping, KANG Hui, YAO Yu-xin, SHEN Tian, CHEN Wei-ping, DU Yuan-peng, GAO Zhen. Back-splicing Primers-based PCR Method for Specific Detection of circRNA[J]. Biotechnology Bulletin, 2022, 38(5): 279-285.

Figures/Tables 10

Table 1 Primers for RT-PCR and qRT-PCR

circRNA名称 Name of circRNA	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
circRNA_4363	AGAGATGCAGAACAACAGAGTTATG	GCGGGATTGAATCTGTGAATC
circRNA_6017	CAGAAGGATACCGTGGAATCGAT	ACTCTGCTGCAGCCTGTAGCAT
circRNA_6044	CATGCAACAGCTTTCAAGCAA	GAGCCTTCCTCCTTTCTTCCA
circRNA_7086	TTTGCTGTTTCTGGATACGCTTCT	TGCAGCATCAACTCTTTCATTGTC

Table 2 Improved primers for RT-PCR and qRT-PCR

circRNA名称 Name of circRNA	正向引物 Forward primer（5'-3'）	反向引物 Reverse primer（5'-3'）
circRNA_4363	ACTGATGGAGTTGATGAGGAA	GGTGTCCTTCTTCGGTTAAACT
circRNA_6017	GCTCTGTAAACTTAACCAGGAAA	GCATGCCATCTGAGATAATCCT
circRNA_6044	AAGAGAGAGCCAAGAAGGAGA	CCTTCTTGCTTGAAAGCTGT
circRNA_7086	CGTTTTTTCTCAGAGAAGAAGG	ATGATGGAAGGATCAGTACCGT

Fig. 1 Alternative back-splicing circularization of circRNA a : Juxtaposition relationship. b : Crossover relationship. c and d : Inclusion relatio-nship

Fig. 2 circRNA produced by exons 7-13 of VIT_201s0010g01480

Fig. 3 RT-PCR results of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086 M: DL2000 marker

Fig. 4 Back-splicing sites and nearby sequences of circRNA

Fig. 5 qRT-PCR dissolution curve of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086

Fig. 6 Improved primer design method

Fig. 7 RT-PCR results of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086 after using improved primers M: DL2000 marker

Fig. 8 qRT-PCR dissolution curve of circRNA_4363, circRNA_6017, circRNA_6044, and circRNA_7086 after using improved primers

References 16

[1]	Cocquerelle C, Mascrez B, Hétuin D, et al. Mis-splicing yields circular RNA molecules[J]. Faseb J, 1993, 7(1):155-160. pmid: 7678559
[2]	Chu QJ, Bai PP, Zhu XT, et al. Characteristics of plant circular RNAs[J]. Brief Bioinform, 2020, 21(1):135-143.
[3]	Tan J, Zhou Z, Niu Y, et al. Identification and functional characterization of tomato CircRNAs derived from genes involved in fruit pigment accumulation[J]. Sci Rep, 2017, 7(1):8594. doi: 10.1038/s41598-017-08806-0 URL
[4]	Lu T, Cui L, Zhou Y, et al. Transcriptome-wide investigation of circular RNAs in rice[J]. RNA, 2015, 21(12):2076-2087. doi: 10.1261/rna.052282.115 URL
[5]	Zeng RF, Zhou JJ, Hu CG, et al. Transcriptome-wide identification and functional prediction of novel and flowering-related circular RNAs from trifoliate orange(Poncirus trifoliata L. Raf. )[J]. Planta, 2018, 247(5):1191-1202. doi: 10.1007/s00425-018-2857-2 URL
[6]	Zheng WQ, Zhang Y, Chen B, et al. Identification and characterization of circRNAs in the developing stem cambium of poplar seedlings[J]. Mol Biol, 2020, 54(5):802-812.
[7]	Ye CY, Zhang X, Chu Q, et al. Full-length sequence assembly reveals circular RNAs with diverse non-GT/AG splicing signals in rice[J]. RNA Biol, 2017, 14(8):1055-1063. doi: 10.1080/15476286.2016.1245268 pmid: 27739910
[8]	Pan T, Sun X, Liu Y, et al. Heat stress alters genome-wide profiles of circular RNAs in Arabidopsis[J]. Plant Mol Biol, 2018, 96(3):217-229. doi: 10.1007/s11103-017-0684-7 URL
[9]	Zhao T, Wang L, Li S, et al. Characterization of conserved circular RNA in polyploid Gossypium species and their ancestors[J]. FEBS Lett, 2017, 591(21):3660-3669. doi: 10.1002/1873-3468.12868 URL
[10]	Tang B, Hao Z, Zhu Y, et al. Genome-wide identification and functional analysis of circRNAs in Zea mays[J]. PLoS One, 2018, 13(12):e0202375. doi: 10.1371/journal.pone.0202375 URL
[11]	高振. 葡萄circRNA鉴定分析及Vv-circATS1的抗冷功能研究[D]. 上海: 上海交通大学, 2019.
	Gao Z. Identification and analysis of grape circRNAs and function research of Vv-circATS 1 on cold resistant[D]. Shanghai: Shanghai Jiaotong University, 2019
[12]	Memczak S, Jens M, Elefsinioti A, et al. Circular RNAs are a large class of animal RNAs with regulatory potency[J]. Nature, 2013, 495(7441):333-338. doi: 10.1038/nature11928 URL
[13]	Gao Y, Wang JF, Zhao FQ. CIRI:an efficient and unbiased algorithm for de novo circular RNA identification[J]. Genome Biol, 2015, 16(1):4. doi: 10.1186/s13059-014-0571-3 URL
[14]	Zhang XO, Wang HB, et al. Complementary sequence-mediated exon circularization[J]. Cell, 2014, 159(1):134-147. doi: 10.1016/j.cell.2014.09.001 URL
[15]	Gao Z, Li J, Luo M, et al. Characterization and cloning of grape circular RNAs identified the cold resistance-related Vv-circATS1[J]. Plant Physiol, 2019, 180(2):966-985. doi: 10.1104/pp.18.01331 URL
[16]	Patop IL, Wüst S, Kadener S. Past, present, and future of circRNAs[J]. EMBO J, 2019, 38(16):e100836.